In this issue, Zhangding et al. developed an experimental technique Repli-MiC and a computational algorithm Fun2 for identifying chromatin fountains associated with DNA replication to investigate transcription-replicatio...In this issue, Zhangding et al. developed an experimental technique Repli-MiC and a computational algorithm Fun2 for identifying chromatin fountains associated with DNA replication to investigate transcription-replication conflicts by measuring the effects of co-directional transcription and head-on transcription on fountains.
Thousands of non-canonical open reading frames (ORFs) in the human transcriptome are translated into microproteins, many with ribosome occupancy comparable to canonical proteins. Intriguingly, most microproteins fail to...Thousands of non-canonical open reading frames (ORFs) in the human transcriptome are translated into microproteins, many with ribosome occupancy comparable to canonical proteins. Intriguingly, most microproteins fail to accumulate as stable proteins; instead, their derived peptides are widely presented by human leukocyte antigen class I (HLA-I) molecules and show emerging immunomodulatory roles. To understand the underlying biology, we explored the folding and stability landscape of a large microprotein cohort, revealing a fundamental rule that connects the genetic code, protein folding, and stability. Structural modeling and parallel profiling revealed that most microproteins are intrinsically disordered and rapidly degraded. Mechanistically, the high GC content of microprotein-coding sequences, which facilitates non-canonical translation, enriches for residues encoded by multiple GC-rich codons (primarily glycine, arginine, alanine, and proline), thereby promoting structural disorder and terminal-residue motif-mediated, Cullin-RING E3 ubiquitin ligase (CRL)-dependent proteasomal degradation. Together, our findings establish a concise, quantitative rule by which high GC content constrains protein evolvability, revealing how surveillance machinery differentially targets microproteins versus canonical proteins.
DNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3' ends. How clamp loaders function at nicks and small single-str...DNA sliding clamps are essential for processive DNA synthesis in all domains of life and are loaded by ATP-dependent clamp loaders that recognize recessed 3' ends. How clamp loaders function at nicks and small single-stranded DNA (ssDNA) gaps-common DNA repair intermediates-remains unclear. Here, we show that the bacterial E. coli DnaX clamp loader uses a mechanism distinct from its eukaryotic counterpart. Whereas eukaryotic replication factor C (RFC) unwinds DNA at the recessed 3' end and stabilizes the 5'-dsDNA (double-stranded DNA) at a shoulder site, the bacterial DnaX-complex neither unwinds DNA nor stably binds the 5'-dsDNA in vitro. Instead, cryo-EM structures reveal that the β-clamp contains a conserved external DNA-binding site that bends gapped DNA by ∼150°, promoting insertion of 3'-dsDNA into the clamp. This DNA-bending mechanism enables efficient β-clamp loading at nicks and small gaps and reveals a distinct bacterial strategy likely important for DNA repair.
Ryan JJ, Miller KR, Puri A
… +14 more, Sprunger ML, Bochantine MM, Lopez A, Chen S, Bao A, Ling C, Xu A, Bell B, Sohn BS, Li L, Chang C, Mohammed K, Staller MV, Jackrel ME
Hsp104, a yeast protein-remodeling factor, can disaggregate misfolded proteins implicated in neurodegeneration. Although many potentiated Hsp104 variants have been generated, suboptimal properties have limited their appl...Hsp104, a yeast protein-remodeling factor, can disaggregate misfolded proteins implicated in neurodegeneration. Although many potentiated Hsp104 variants have been generated, suboptimal properties have limited their application in mammalian systems. Here, we present the development of a high-throughput screening approach for identifying enhanced Hsp104 variants. To screen a large library of variants in parallel and with a quantitative output, we coupled a live-or-die yeast-based selection with next-generation sequencing. The identified Hsp104 variants solubilize preformed α-synuclein and TDP-43 aggregates, inhibit seeding of preformed α-synuclein fibrils in mammalian biosensor cells, restore TDP-43 splicing of native targets, and have diminished off-target toxicity in mammalian cells. Certain variants show distinct changes in ATP hydrolysis, which we suggest is the key driver of these improved properties. We anticipate that our approach is broadly applicable to a range of protein engineering targets to allow coupling of a phenotypic readout to high-throughput quantitative analysis of variants in parallel.
Karnay A, Linares-Saldana R, Wang Q
… +18 more, Gardner Z, Liang JA, Santini GT, Haridhasapavalan KK, Nguyen SC, Hugelier S, Shewale B, Kanemaki MT, Napierala JS, Napierala M, Wilson RB, Dubois N, Poleshko A, Kim W, Shah PP, Lakadamyali M, Joyce EF, Jain R
Variability in genome organization drives differential gene expression and shapes cellular diversity, yet whether transcription actively instructs genome structure and how this relationship is exploited in disease remain...Variability in genome organization drives differential gene expression and shapes cellular diversity, yet whether transcription actively instructs genome structure and how this relationship is exploited in disease remains unclear. We show that transcription and cohesin direct the spatial positioning of lamina-associated domain (LAD) boundary genes. Transcriptional repression repositions LAD boundary genes to the nuclear lamina in a cohesin loop extrusion-dependent manner. Conversely, overactive cohesin is sufficient to reposition and silence LAD boundary genes, an effect counteracted by maintaining transcription. In Friedreich's ataxia, we demonstrate improper positioning of the pathogenically repressed LAD boundary gene FRATAXIN (FXN) at the nuclear periphery reflects an imbalance between transcription and cohesin dynamics. Importantly, modulating either transcription or cohesin activity restores FXN positioning and reactivates expression. Our findings establish transcription and cohesin as tunable molecular rheostats orchestrating LAD boundary spatial positioning and reveal how the flexible and dynamic nature of genome architecture is hijacked in disease.
TDP43 inclusion bodies are widely present in the majority of patients with familial and sporadic amyotrophic lateral sclerosis (ALS). The mechanisms regulating TDP43 solubility remain incompletely understood. Here, we re...TDP43 inclusion bodies are widely present in the majority of patients with familial and sporadic amyotrophic lateral sclerosis (ALS). The mechanisms regulating TDP43 solubility remain incompletely understood. Here, we report that TDP43 undergoes S-acylation primarily at the Cys244 residue by the S-acyltransferase zDHHC23. This S-acylation maintains the liquid-like properties of TDP43 by reducing the aberrant interaction with poly(ADP-ribose) polymerase 1 (PARP1) and PARylated proteins, thereby countering the pathological condensation of TDP43. S-acylation-deficient TDP43 inclusions sequester the translational machinery and inhibit cytoplasmic protein translation, ultimately resulting in neurotoxicity. Importantly, TDP43 S-acylation is decreased in the familial ALS-associated TDP43 mutants as well as in SOD1-G93A mice and C9orf72-ALS induced pluripotent stem cell (iPSC)-derived neurons, suggesting the widespread involvement of TDP43 S-acylation in ALS pathogenesis. Our findings reveal an undescribed modification of TDP43 and provide deeper insight into the regulation of TDP43 pathological condensation in ALS.
Protein interactions with canonical B-form DNA are well characterized, yet the effect of torsionally constrained DNA on these interactions-ubiquitous in cells-remains underexplored. Using cryo-electron tomography (cryo-E...Protein interactions with canonical B-form DNA are well characterized, yet the effect of torsionally constrained DNA on these interactions-ubiquitous in cells-remains underexplored. Using cryo-electron tomography (cryo-ET), we 3D-reconstructed entire negatively supercoiled DNA substrates bound to active RNA polymerase (RNAP), revealing diverse DNA supercoiling conformations and their interplay with transcription. RNAP preferentially localizes at plectoneme apices in a swiveled, pause-prone state. RNAP, along with other DNA-melting proteins such as dCas9, can act as torsional roadblocks that segregate "twin-supercoiling domains" during active transcription, independent of external DNA/RNAP tethering. Co-transcribing RNAPs further intensify this domain separation: tandem-oriented RNAPs relieve negative supercoiling more effectively than opposing ones and promote greater RNAP accumulation and enhanced elongation, both in vitro and in vivo. Topoisomerase I relieves torsional stress and facilitates RNAP escape from apical stalls, thereby supporting apical transcription regulation. Together, these findings support a load-and-release mechanism at plectoneme apices that may underlie supercoiling-dependent transcriptional bursting.
Wolczyk M, Szymanski J, Trus I
… +13 more, Naz Z, Idlin N, Lechowski M, Baranowska E, Bis A, Nowak JS, Tame T, Jackiewicz J, Nowak E, Wuebben C, Hartmann G, Rappsilber J, Michlewski G
The interferon (IFN) response is crucial for antiviral activity, but excessive activation can contribute to tissue damage and autoimmune disorders. The cytoplasmic receptor retinoic acid-inducible gene I (RIG-I) detects...The interferon (IFN) response is crucial for antiviral activity, but excessive activation can contribute to tissue damage and autoimmune disorders. The cytoplasmic receptor retinoic acid-inducible gene I (RIG-I) detects viral double-stranded RNAs (dsRNAs) and endogenous RNA polymerase III (RNA Pol III) transcripts carrying a 5'-triphosphate (5'-ppp) moiety, triggering IRF3 phosphorylation and IFN response. Many viral RNAs initiate with 5'-triphosphate adenosine (5'-pppA), whereas most endogenous RNA Pol III transcripts in higher eukaryotes start with 5'-triphosphate guanosine (5'-pppG), yet no reason for this bias is known. Here, we show that 5'-pppA dsRNAs trigger stronger RIG-I/IFN responses than 5'-pppG dsRNAs, despite comparable RIG-I stimulation in vitro. Using RNA pull-down and mass spectrometry, we identify GTP-binding proteins that preferentially interact with 5'-pppG dsRNAs. Guanosine supplementation, which increases intracellular GTP levels, decreases the immunogenic difference between 5'-pppG and 5'-pppA dsRNAs. Our findings provide insights into sequence-dependent activation of the RIG-I/IFN pathway, with implications for evolutionary pressures on RNA sequences, RNA-driven autoimmunity, viral immunogenicity, and the rational design of RNA therapeutics.
Diaz-Del-Olmo I, O'Sullivan PA, Wilson TS
… +10 more, Majstorovic A, Miller G, Shizukuishi S, Stypulkowska A, Panagi I, Grzymajlo K, Ogawa M, Bezbradica JS, Hill PWS, Thurston TLM
The ability of Salmonella Typhimurium to exploit macrophages as a niche for survival, replication, and dissemination is central to its pathogenesis. The effector SteE, which polarizes macrophages into an anti-inflammator...The ability of Salmonella Typhimurium to exploit macrophages as a niche for survival, replication, and dissemination is central to its pathogenesis. The effector SteE, which polarizes macrophages into an anti-inflammatory state, is critical during invasive disease. SteE operates via an unprecedented mechanism, reprogramming the host serine/threonine kinase GSK3 to perform tyrosyl-directed phosphorylation of neosubstrates, including the immune transcription factors STAT1 and STAT3. Here, we demonstrate that SteE-driven transcriptional reprogramming relies critically and specifically on STAT3 phosphorylation and DNA binding. By activating STAT3 via a non-canonical pathway, bypassing endogenous negative feedback mechanisms, SteE drives hyperactivation of STAT3 target genes, surpassing the effects of canonical IL-10 signaling. Hyperactivation correlates with elevated phosphorylated STAT3 in the macrophage nucleus and coordinated chromatin remodeling at STAT3 target loci. Overall, our study illustrates how hijacking of a signaling pathway by SteE dramatically reshapes the macrophage gene regulatory network to enhance Salmonella immune evasion.
In a recent issue of Cell, Xie et al. report that an important function of mitochondrial aconitase is to limit toxic citrate accumulation, suggesting a role for the canonical TCA cycle in physiology beyond ATP production...In a recent issue of Cell, Xie et al. report that an important function of mitochondrial aconitase is to limit toxic citrate accumulation, suggesting a role for the canonical TCA cycle in physiology beyond ATP production and precursor biosynthesis.
Extracellular alkalinization is a hallmark of plant immunity. In this issue of Molecular Cell, Zhai et al. and in a recent issue of Cell, Wang et al. reveal that extracellular alkalinization is regulated by phosphorylati...Extracellular alkalinization is a hallmark of plant immunity. In this issue of Molecular Cell, Zhai et al. and in a recent issue of Cell, Wang et al. reveal that extracellular alkalinization is regulated by phosphorylation of proton pumps, which is crucial for local defense and systemic immune priming.
In this issue, Losa et al. identify agglomerates composed of enzymes involved in amino acid metabolism as important factors controlling cytoplasmic fluidity in bacteria that are dependent on growth conditions. The obstac...In this issue, Losa et al. identify agglomerates composed of enzymes involved in amino acid metabolism as important factors controlling cytoplasmic fluidity in bacteria that are dependent on growth conditions. The obstacles hinder the diffusion of large, ribosome-sized objects, while freeing space for faster diffusion of smaller proteins.
In this issue of Molecular Cell, Zaratiana and colleagues have evaluated the activity of cis-regulatory elements (CREs) by combining massively parallel reporter assays (MPRAs) and microbial perturbation experiments. The...In this issue of Molecular Cell, Zaratiana and colleagues have evaluated the activity of cis-regulatory elements (CREs) by combining massively parallel reporter assays (MPRAs) and microbial perturbation experiments. The work uncovers a direct link between microbial metabolites and the regulatory DNA sequences that control hepatic transcription.
Ahmed B, Salaun D, Jordan JB
… +18 more, Daulat A, Pastorello C, Brito T, Yang Y, Josselin E, Byrne D, Betzi S, Pelletier A, Vernerey J, Ferreira L, Castellano R, Audebert S, Camoin L, Pane A, Bechara C, Borg JP, Modesti M, Lignitto L
Lung cancers frequently increase iron demand to sustain growth, which makes them vulnerable to ferroptosis. While Cullin 2-RING ubiquitin ligases (CRL2s) are critical regulators of stress responses and redox balance, the...Lung cancers frequently increase iron demand to sustain growth, which makes them vulnerable to ferroptosis. While Cullin 2-RING ubiquitin ligases (CRL2s) are critical regulators of stress responses and redox balance, their role in ferroptosis mechanisms remains largely unknown. Here, we identify the E3 ligase CRL2 as a key regulator of the ferroptotic response. CRL2 recruits BTB and CNC homolog 1 (BACH1), a transcriptional regulator of ferroptosis, for degradation by recognizing a degron that is directly formed by the redox-sensing molecule heme. By degrading BACH1 in response to heme, CRL2 acts as a switch that dynamically modulates the transcriptional activation of ferroptosis-protective genes, particularly solute carrier family 7 member 11 (SLC7A11). Loss of CRL2 stabilizes BACH1 and suppresses SLC7A11, thereby sensitizing lung tumor cells to ferroptosis inducers in vitro and in preclinical models. Our findings identify CRL2 as a target to increase the efficacy of ferroptosis inducers in lung cancer treatment and define a broader principle whereby endogenous metabolites regulate protein degradation by enabling substrate-E3 ligase interactions.
Cheng P, Mermerian-Baghdassarian A, Wang Y
… +13 more, Chen Z, Quysbertf HM, Cheema PS, Mays JC, Zhao X, Katsnelson L, Mei S, Shrivastava R, Bulatovic M, Deng J, Schober M, Wong KK, Davoli T
Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistan...Aneuploidy is common in cancer and has been implicated in promoting tumor progression, yet the underlying mechanisms remain poorly understood. By generating models of aneuploidy, we found that aneuploidy confers resistance to reactive oxygen species (ROS)-mediated cell death, independent of the specific chromosomes gained or lost. Mechanistically, poly(ADP-ribose) polymerase 1 (PARP1) is suppressed in aneuploid cells, which inhibits PARP1-mediated cell death (parthanatos). We validated aneuploidy-associated PARP1 suppression across 15 cell models and human tumors, with pronounced effects in metastatic tumors. Importantly, PARP1 downregulation promotes tumor metastasis while PARP1 upregulation suppresses it. Through a genome-wide CRISPR screen and functional validation, we identified the transcription factor CCAAT/enhancer-binding protein beta (CEBPB) as a mediator of PARP1 downregulation and ROS resistance in aneuploid cells. Lysosomal dysfunction serves as the upstream activator of CEBPB in aneuploid cells. We propose that aneuploidy-driven CEBPB activation suppresses PARP1, fostering ROS resistance and cancer progression.
RNA polymerase II (RNA Pol II) is central to gene expression, but its catalytic mechanism remains elusive due to the absence of high-resolution structural data. The role of water molecules in RNA Pol II catalysis is unkn...RNA polymerase II (RNA Pol II) is central to gene expression, but its catalytic mechanism remains elusive due to the absence of high-resolution structural data. The role of water molecules in RNA Pol II catalysis is unknown. Here, we present 3 high-resolution cryo-electron microscopy structures of active Saccharomyces cerevisiae RNA Pol II elongation complexes in distinct catalytic states: two pre-catalysis states at 1.96 Å and 2.26 Å resolution and a post-catalysis state at 2.33 Å resolution. Each structure contains over 700-1,350 ordered water molecules, many located at functionally critical positions. Comparative analysis shows that these waters play essential roles in proton-transfer steps during RNA Pol II catalysis, facilitating substrate recognition and trigger-loop folding during nucleotide addition. Strikingly, these waters are conserved between prokaryotic and eukaryotic transcription machineries (see Mueller and Darst). These findings provide unprecedented mechanistic insights into RNA Pol II catalysis and reveal vital and evolutionarily conserved roles of water molecules in transcription.
Multi-subunit DNA-dependent RNA polymerase (RNAP) is the central enzyme of transcription, yet its catalytic mechanism remains obscure because high-resolution structures of intermediates with native substrates are not ava...Multi-subunit DNA-dependent RNA polymerase (RNAP) is the central enzyme of transcription, yet its catalytic mechanism remains obscure because high-resolution structures of intermediates with native substrates are not available. We visualized E. coli RNAP on a promoter DNA template with nucleoside triphosphate substrates engaged in active RNA synthesis by cryo-electron microscopy. From this heterogeneous mixture, we determined five high-resolution structures of initial transcribing complexes, including a true Michaelis complex (MC) and a post-catalytic product complex (PC). The MC reveals key conformational transitions during catalysis. Waters in the MC and PC structures show striking overlap with those in corresponding S. cerevisiae RNA polymerase II (RNAPII) structures (see related paper by Li et al.), pointing to functional importance. Together, these results establish that RNAP catalyzes nucleotidyl transfer through a positional (entropic) mechanism, revealing structural determinants of the first step of gene expression.
Proteotoxic stress, characterized by the accumulation of damaged proteins, poses a significant challenge to cellular homeostasis. To mitigate proteotoxicity, eukaryotes rely on the proteasome, which is regulated by prote...Proteotoxic stress, characterized by the accumulation of damaged proteins, poses a significant challenge to cellular homeostasis. To mitigate proteotoxicity, eukaryotes rely on the proteasome, which is regulated by proteasome transcriptional activators. As proteotoxicity can originate in different compartments, cells must integrate signals from multiple locations, yet the mechanisms coordinating this response remain unclear. Here, we show that the proteasome autoregulatory feedback loop functions as a gatekeeper enabling the communication between the nucleus and chloroplast. At the endoplasmic reticulum (ER), the plant proteasome transcriptional activators NAC53 and NAC78 undergo either ER-associated degradation (ERAD) or are released for nuclear translocation. We define this dual mechanism as ER-associated sorting (ERAS). While NAC53/78 activate proteasome gene expression, they repress photosynthesis-associated nuclear genes during proteotoxicity through a conserved cis-element. Together, our findings reveal a trade-off between proteasome activity and energy metabolism and establish a framework in which the proteasome feedback loop coordinates subcellular proteostasis and growth-defense balance.
Extrachromosomal circular DNA (eccDNA) drives genome instability and tumorigenesis, warranting thorough investigations of its biogenesis. Here, we report a mechanism of eccDNA formation in human cells, distinct from the...Extrachromosomal circular DNA (eccDNA) drives genome instability and tumorigenesis, warranting thorough investigations of its biogenesis. Here, we report a mechanism of eccDNA formation in human cells, distinct from the one mediated by joining of broken DNA ends. We show that repeats such as telomeres and centromeric alpha satellite form internal loops (i-loops) as a consequence of single-stranded DNA (ssDNA) breaks or gaps rather than double-stranded breaks (DSBs). I-loops are precursors for the excision of eccDNA, visible by electron microscopy (EM) and detectable via rolling-circle amplification. Apoptosis triggers the formation of i-loops and eccDNA at telomeric and alpha satellite repeats. Nanopore sequencing revealed other repetitive elements, including rDNA and retrotransposons, as sources of eccDNA in apoptosis. Based on the prevalence of SSBs over DSBs and the abundance of repeats in the human genome, we propose that the i-loop mechanism contributes substantially to all forms of eccDNA, with implications for tumor biology and genome evolution.
MYCN-amplified neuroblastoma is one of the most lethal pediatric malignancies, where aberrant N-Myc-driven transcription promotes tumor progression. As direct targeting of N-Myc has proven challenging, current approaches...MYCN-amplified neuroblastoma is one of the most lethal pediatric malignancies, where aberrant N-Myc-driven transcription promotes tumor progression. As direct targeting of N-Myc has proven challenging, current approaches prioritize understanding the mechanisms that regulate its activity, which remain poorly understood. Here, we demonstrate a crucial role of S-acylation in regulating N-Myc transcriptional activity and identify the acyltransferase zinc finger DHHC-type containing 22 (ZDHHC22) as a key regulator of this process. Mechanistically, ZDHHC22 catalyzes the S-acylation of N-Myc, which enhances its transcriptional activity by facilitating the recruitment of coactivators such as TIP60 and GCN5. Furthermore, N-Myc transcriptionally upregulates ZDHHC22, establishing a feedback loop that contributes to chemoresistance in high-risk neuroblastoma. Targeting ZDHHC22 suppresses neuroblastoma cell growth in vitro and in vivo, particularly in refractory patient-derived models. Collectively, our findings uncover a biological function of ZDHHC22 in regulating N-Myc transcriptional activation and indicate that ZDHHC22 is a promising therapeutic target for N-Myc-driven high-risk neuroblastoma, especially in MYCN-amplified patients.